SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets. Although SADS-CoV uses different strategies to evade the host's innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified. In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected. The results determined that nsp1 was a potent antagonist of IFN response. SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation. Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin–proteasome pathway. Furthermore, SADS-CoV nsp1 induced CREB-binding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling. In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK–STAT signaling pathway via the ubiquitin–proteasome pathway. This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK–STAT signaling pathway and the strategies of SADS-CoV in evading the host's innate immune system. The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets. Although SADS-CoV uses different strategies to evade the host's innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified. In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected. The results determined that nsp1 was a potent antagonist of IFN response. SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation. Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin–proteasome pathway. Furthermore, SADS-CoV nsp1 induced CREB-binding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling. In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK–STAT signaling pathway via the ubiquitin–proteasome pathway. This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK–STAT signaling pathway and the strategies of SADS-CoV in evading the host's innate immune system. Coronaviruses have significantly challenged worldwide public health in the last 20 years. Coronaviruses are frequently present in animal and human groups and can cause economic disruption and catastrophic loss of life (1Ellwanger J.H. Chies J.A.B. Zoonotic spillover: understanding basic aspects for better prevention.Genet. Mol. Biol. 2021; 44e20200355Crossref Google Scholar). In February 2017, the newly discovered swine acute diarrhea syndrome coronavirus (SADS-CoV) was first reported in southern China. SADS-CoV infection leads to acute diarrhea, vomiting, and high mortality rates among young piglets (particularly those <7 days old) but only causes mild or asymptomatic infections in adult swine (2Zhou L. Sun Y. Lan T. Wu R. Chen J. 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AIP4/Itch regulates Notch receptor degradation in the absence of ligand.PLoS One. 2008; 3e2735Crossref PubMed Scopus (111) Google Scholar). Additionally, only K63-linked Ub chains do not target proteins for proteasomal degradation (31Rabl J. BRCA1-A and BRISC: multifunctional molecular machines for ubiquitin signaling.Biomolecules. 2020; 10: 1503Crossref PubMed Scopus (14) Google Scholar). Several studies provided evidence of the capacity of different coronaviruses to block IFN production and response by specifically targeting and degrading the host's antiviral elements via the Ub-proteasome pathway. The severe actuate respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 promotes the degradation of CHK1, a kinase involved in responding to DNA damage, through the proteasome pathway (35Gioia U. Tavella S. Martínez-Orellana P. Cicio G. Colliva A. Ceccon M. et al.SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.Nat. Cell Biol. 2023; 25: 550-564Crossref PubMed Scopus (19) Google Scholar). SADS-CoV N protein interacts with RIG-I and induces its degradation through the Ub–proteasome pathway (36Liu Y. Liang Q.Z. Lu W. Yang Y.L. Chen R. Huang Y.W. et al.A comparative analysis of coronavirus nucleocapsid (N) proteins reveals the SADS-CoV N protein antagonizes IFN-β production by inducing ubiquitination of RIG-I.Front Immunol. 2021; 12688758Google Scholar). No research to date has indicated the inhibition of IFN response by SADS-CoV nonstructural proteins through hijacking the Ub-proteasome system (UPS). Previously, our study demonstrated that SADS-CoV nsp1 significantly inhibits IFN production (37Xiang Y. Mou C. Shi K. Chen X. Meng X. Bao W. et al.SADS-CoV nsp1 inhibits the IFN-β production by preventing TBK1 phosphorylation and inducing CBP degradation.J. Med. Virol. 2023; 95e29104Crossref Scopus (2) Google Scholar). This inhibition was primarily attributed to nsp1 blocking of TBK1 Ub-like modification, which suppressed TBK1 phosphorylation. Furthermore, nsp1 induces CBP degradation through the Ub–proteasome pathway, thereby blocking IFN transcription enhancer formation. In this study, the potential of SADS-CoV nonstructural proteins to inhibit the IFN response was detected. SADS-CoV nsp1 was eventually identified as a potent antagonist in the IFN response. Nsp1 was important in IFN inhibition by SADS-CoV. This inhibition promoted SADS-CoV replication during the early stages of viral infection. Despite earlier studies also demonstrated that coronavirus nsp1 blocks the JAK-STAT signaling pathway, the potential mechanism remains unclear. Our research indicated that SADS-CoV nsp1 inhibited the JAK-STAT signaling pathway by blocking STAT1 phosphorylation and acetylation. This blocking was attributed to the nsp1-induced JAK1 and CBP degradation. Overall, the investigation revealed new mechanisms by which SADS-CoV nsp1 blocked the IFN response. The findings contribute to comprehending the strategies utilized by coronavirus nsp1 to evade the host innate immune system. Previously, the authors demonstrated that the Phe39 and Leu98 of SADS-CoV nsp1 were critical amino acids in antagonizing IFN production. These SADS-CoV nsp1 amino acids were mutated, ultimately generating a mutant virus (SADS-CoV-mutant) (Fig. 1A). Then, whether SADS-CoV-mutant infection inhibited IFN production and response was investigated. LLC-PK1 cells were infected with SADS-CoV-WT (wildtype) or SADS-CoV-mutant, and the cells were collected at 4, 6, and 8 h postinfection (hpi). The IFN-β and ISG15 mRNA levels were significantly increased in cells infected with SADS-CoV-mutant compared to the cells infected with SADS-CoV-WT. This result indicated that the SADS-CoV-mutant was not able to effectively inhibit IFN production and response (Fig. 1, B and C). To determine if the ability of nsp1 to block the IFN system is essential for SADS-CoV replication, the SADS-CoV-WT and SADS-CoV-mutant growth kinetics were compared via a growth curve assay. In ST cells and Vero cells, the SADS-CoV-mutant produced lower quantities of progeny virions compared to SADS-CoV-WT from 8 hpi to 24 hpi. Due to the absence of IFN genes in Vero cells, this difference is more significant in ST cells than in Vero cells. (Fig. 1, D and E). The immunofluorescence assay result demonstrated that the proportion of cells infected with SADS-CoV-mutant was significantly reduced compared to cells infected with SADS-CoV-WT at 12 hpi (Fig. 1F). These results suggested that nsp1 was critical in SADS-CoV antagonism of IFN production and response. Additionally, the nsp1 mutations reduced the SADS-CoV replication capacity. STAT1 and STAT2 combine to form ISGF3 with IRF9 and subsequently induce ISRE-driven ISG transcription in the nucleus. Whether nsp1 inhibited the ISRE promoter activity stimulated by STAT1, STAT2, IRF9, or ISGF3 was detected to explore the potential regulatory role of SADS-CoV nsp1 in the IFN response. The ISRE promoter activity was examined using a dual-luciferase reporter assay, whereas the exogenous protein expression was detected via Western blotting. Compared to the negative control, STAT1, STAT2, IRF9, ISGF3, and human IFN-β significantly induced ISRE promoter activation. Contrastingly, nsp1 significantly inhibited ISRE promoter activity in cells stimulated by human IFN-β, STAT1, STAT2, or IRF9 (Fig. 2, A–C and E). However, this inhibition was attenuated in the ISFG3-stimulated cells, suggesting that nsp1 blocked the IFN response by targeting ISGF3 complex formation (Fig. 2D). Furthermore, nsp1 had no negative effect on exogenous STAT1, STAT2, IRF9, and ISGF3 expression levels (Fig. 2F). The suppressive effect of nsp1 on ISG15, ISG56, and 2′-5′-oligoadenylate synthetase 1 (OAS1) mRNA levels was investigated. Real-time PCR analyses revealed that nsp1 efficiently inhibited ISG15, ISG56, and OAS1 expression in cells stimulated by human IFN-β, STAT1, STAT2, or IRF9 (Fig. 2, G–I). Furthermore, Phe39 and Lys98 in the nsp1 sequence were mutated to alanine, generating a nsp1-mutant. Compared with the nsp1-transfected cells, the ISG15, ISG56, and OAS1 mRNA levels were not significantly inhibited in the cells transfected with the nsp1-mutant. This result suggested that Phe39 and Lys98 mutation might lead to the loss of nsp1 function in antagonizing the IFN response (Fig. 2J). These findings indicated that SADS-CoV nsp1 strongly inhibited the IFN response. STAT1 and STAT2 are crucial transcription activators in the JAK–STAT pathway. Endogenous STAT1 and STAT2 expression and phosphorylation were detected by Western blotting to investigate the effect of nsp1 and nsp1-mutant on these proteins. The result demonstrated that nsp1 decreased STAT1 phosphorylation in human embryonic kidney cell line (HEK)-293T cells stimulated by human IFN-β and LLC-PK1 cells induced by Sendai virus (SeV). The nsp1-mutant did not show a similar function (Fig. 3, A–C). Nevertheless, nsp1 did not block STAT2 phosphorylation. Nsp1 expression did not influence STAT1 and STAT2 endogenous expression, suggesting that nsp1 specifically inhibited STAT1 phosphorylation (Fig. 3, A and C). Furthermore, nsp1 induced IRF9 degradation (data not shown). These results indicated that STAT1 and IRF9 are the primary targets of nsp1-mediated inhibition of the IFN response. After observing the nsp1-induced inhibition of STAT1 phosphorylation, the investigation was subsequently focused on the endogenous expression and phosphorylation of JAK1 and TYK2, which are crucial kinases for inducing STAT1 activation. Nsp1 efficiently induced JAK1 degradation in HEK-293 cells and LLC-PK1 cells, whereas the nsp1-mutant did not induce the JAK1 degradation (Fig. 4, A–D). Contrastingly, endogenous TYK2 expression and phosphorylation were not inhibited in the nsp1-expressing cells (Fig. 4, A and B). The results demonstrated that nsp1 might degrade JAK1 to inhibit STAT1 phosphorylation, thereby blocking the JAK-STAT signaling pathway. The pathway of JAK1 degradation by nsp1 was analyzed to investigate the mechanism through which nsp1 downregulated JAK1 expression. Specifically, HEK-293T cells were treated with Dulbecco's modified Eagle's medium (DMEM, the negative control), 10 μM MG132 (a proteasome pathway inhibitor), 5 μM Z-VAD-FMK (an apoptosis pathway inhibitor), or 10 μg/ml NH4Cl (an autophagy pathway inhibitor). Nsp1 persistently induced the degradation of endogenous JAK1 in all treated cells (Fig. 5, A–C). However, this degradation was effectively attenuated in cells treated with 5 μM MG132, suggesting that MG132 is an effective antagonist against nsp1-induced JAK1 degradation (Fig. 5D). These results indicated that nsp1 degraded JAK1 through the proteasome pathway. Subsequently, it was detected that nsp1 inhibited the STAT1 phosphorylation level in MG132-treated cells. Compared to the negative control, nsp1 did not induce JAK1 degradation and inhibit STAT1 phosphorylation (Fig. 5E). Overall, the results indicated that nsp1 inhibited STAT1 phosphorylation by inducing JAK1 degradation via the proteasome pathway. Protein degradation mediated by the Ub-proteasome pathway can occur via monoubiquitination or polyubiquitination. HEK-293T cells were transfected with Ub or Ub mutants (Ub-K6R, Ub-K11R, Ub-27R, Ub-K29R, Ub-K33R, Ub-K48R, and Ub-K63R) to investigate the specific form of nsp1-induced JAK1 ubiquitination. The Ub mutants were characterized by replacing the lysine residues (K6, K11, K27, K29, K33, K48, or K63) with arginine. After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1. Then, the cells were collected to detect the interaction between JAK1 and Ub (or Ub mutants) by Western blotting. Nsp1 induced the interaction between JAK1 and Ub (Fig. 6A). Furthermore, nsp1 promoted interaction between JAK1 and Ub-K6R, Ub-K27R, Ub-K29R, and Ub-K63R, indicating that silencing K6, K27R, K29, and K63 of Ub did not significantly impact JAK1 degradation (Fig. 6, B, D–F and H). Conversely, nsp1 did not induce interaction between JAK1 and Ub-K11R and Ub-K48R, suggesting that K11 and K48 were critical lysines in nsp1-induced JAK1 degradation (Fig. 6, C and G). The above results were validated by detecting the interaction between JAK1 and other Ub mutants, including Ub-KO (a mutated type of Ub where all seven lysine residues are replaced with arginine), Ub-K6O (a mutated type of Ub where all lysine residues except lysine 6 are replaced with arginine), Ub-K11O, Ub-K27O, Ub-K29O, Ub-K33O, Ub-K48O, and Ub-K63O. Nsp1 induced JAK1 ubiquitination in the cells expressing Ub-K11O and Ub-K48O (Fig. 7, C and G). Additionally, nsp1 did not induce the interaction between JAK1 and Ub-KO, Ub-K6O, Ub-K27O, Ub-K29O, and Ub-K63O (Fig. 7, A, B, D–F and H). These results indicated that nsp1 degraded JAK1 through K11 and K48-linked polyubiquitination. In the nucleus, p-STAT1 interacts with CBP to induce ISG transcription and undergoes acetylation. The acetylated STAT1 binds TCPTP to undergo dephosphorylation. HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1 and stimulated by human IFN-β to detect whether nsp1 inhibited STAT1 acetylation and dephosphorylation. The cells were collected to detect STAT1 acetylation and the interaction between STAT1 and TCPTP. Nsp1 significantly inhibited STAT1 acetylation (Fig. 8A). Based on previous findings on nsp1-induced CBP degradation, it was concluded that nsp1 inhibited STAT1 acetylation by degrading CBP. Furthermore, nsp1 did not affect TCPTP expression. However, it slightly inhibited the interaction between STAT1 and TCPTP, suggesting that nsp1 blocked STAT1 dephosphorylation by inhibiting STAT1 acetylation (Fig. 8A). To verify these results, the cells were treated with MG132 to eliminate the nsp1 function in degrading CBP, and the STAT1 acetylation and dephosphorylation were detected. The nsp1 inhibition of STAT1 acetylation and dephosphorylation was significantly attenuated when CBP was no longer degraded (Fig. 8B). Additionally, nsp1-mutant did not inhibit STAT1 acetylation and dephosphorylation (Fig. 8C). Nsp1 inhibition of STAT1 acetylation might block STAT1 export from the nucleus to the cytoplasm. HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1 to verify this finding. At 24 h or 36 h post-transfection, the cells underwent nucleocytoplasmic fractionation. Nsp1 significantly degraded CBP in both the 24-h and 36-h groups (Fig. 8, D and E). In the 24-h group, nsp1 increased STAT1 content in the cytoplasm and decreased p-STAT1 content in the nucleus by inhibiting STAT1 phosphorylation. In the 36-h group, STAT1 content in the cytoplasm was decreased, and p-STAT1 content in the nucleus was increased, which was due to nsp1 blocking STAT1 acetylation. These results suggested that nsp1 blocked STAT1 nuclear translocation by inhibiting STAT1 phosphorylation and prevented STAT1 translocation out of the nucleus by inhibiting STAT1 acetylation. Host cells adopt multiple strategies to defend against coronaviruses, including the IFN response. Simultaneously, coronaviruses have developed various methods to evade the host's innate immune response by blocking the IFN response. Increasing evidence indicates that coronavirus nonstructural proteins are important in suppressing the IFN response by targeting the JAK-STAT signaling pathway. For example, porcine deltacoronavirus nsp5 cleaves STAT2 to block ISGF3 formation (38Zhu X. Wang D. Zhou J. Pan T. Chen J. Yang Y. et al.Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving STAT2.J. Virol. 2017; 91e00003-17Crossref Scopus (107) Google Scholar). SARS-CoV-2 nsp13 and ORF6 block STAT1 nuclear translocation to inhibit ISG transcription (39Yuen C.K. Lam J.Y. Wong W.M. 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